5-Oxo-L-prolinase (5-OPase) is involved in the synthesis and metabolism of glutathione (GSH) in the gamma-glutamyl cycle. The present project is to investigate (1) the level and distribution of 5-OPase in human tissues, (2) the potential application of a 5-OPase modulator, L-2-oxothiazolidine-4-carboxylate (OTZ), in selective modulation of GSH in normal versus tumor cells in cancer chemotherapy, and (3) the role of 5-OPase in cellular GSH modulation. / First, Compared to rat tissues, 5-OPase activity was found to be generally low in human tissues and no specific distribution pattern was observed. A polyclonal rabbit anti-rat 5-OPase antibody was produced with high affinity and specificity and it was shown to crossreact with human 5-OPase. Immunohistochemistry analysis revealed that 5-OPase was localized in the structures that have typical absorptive or secretary function. By Western blot, normal human lung and stomach tissues showed significantly higher 5-OPase levels than their paired tumors, while the other tissues examined showed case-dependent patterns. / Second, OTZ, a 5-oxo-L-proline analog that is metabolized to cysteine by 5-OPase, was shown in vivo to increase the GSH level in normal bone marrow cells while paradoxically decreasing it in rat mammary tumors and sensitizing the tumors to the alkylating agent melphalan. When the human breast cancer cell line MCF7 was treated with OTZ in combination with melphalan, the cytotoxicity of melphalan was increased relative to that of melphalan alone, and this effect was reversed by the addition of glutamate or 5-oxo-L-proline. OTZ treatment decreased the GSH levels in MCF7 cells but increased the GSH levels in normal human breast MCF10A cells. Therefore, in tumor cells where glutamate is limited in GSH synthesis, OTZ increases melphalan toxicity by limiting glutamate production from 5-OPase for GSH synthesis. / Third, the expression of transfected 5-OPase in MCF7 cells decreased the cellular GSH level, sensitized the Cells to melphalan toxicity, and diminished the sensitizing effect of OTZ on melphalan toxicity; while exposure of both normal and cancerous human cells to GSH depleting agents led to an increased expression of 5-OPase both in vitro and in vivo. These results indicate a critical interaction between cellular GSH levels and 5-OPase activity that could be important in GSH modulation in therapeutic settings.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.36562 |
| Date | January 1999 |
| Creators | Chen, Xiang, 1968- |
| Contributors | Batist, Gerald (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Division of Experimental Medicine.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001740507, proquestno: NQ64533, Theses scanned by UMI/ProQuest. |
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